The objective of our laboratory is to understand the molecular determinants of cellular survival that allow tumor cells to escape programmed cell death (apoptosis) when they are exposed to chemotherapy or irradiation. Identifying specific molecules that promote survival will provide new, attractive targets for the development of compounds that abrogate survival signals and enhance therapeutic effectiveness. Cellular survival is determined by factors both within the cell and outside the cell, including the contribution of extracellular influences such as soluble growth factors and extracellular matrix molecules. Both growth factors and extracellular matrix molecules stimulate survival through activation of enzymatic pathways within the cell that involve proteins that either add phosphate to downstream substrates (kinase) or remove phosphate (phosphatases). The best described survival pathways depend on activity from kinases such as P13K, Akt, PKC, and MAPK, that become activated when they themselves are phosphorylated. Activation can occur after binding of extracellular growth factors to their cognate receptors, or in the case of some tumor cells, activation is independent of extracellular growth factors and is constitutive. Recently, we have described a role for three signaling pathways that contribute to the survival and therapeutic resistance of lung cancer cells: the PI3K/Akt pathway, the MEK/ERK pathway, and pathways involving isoforms of PKC. Because lung cancer cells appear to be most dependent upon the Akt pathway, we have focused in the last year on this pathway. Four separate studies from our group have highlighted the importance of the PI3K/Akt/mTOR pathway. First, tobacco components activate the pathway in two types of normal human lung epithelial cells, which causes an Akt-dependent, partially transformed phenotype. Second, lung lesions induced by a tobacco carcinogen, NNK, are characterized by progressive activation of the Akt pathway. Third, most lung cancer cells have constitutively activated Akt, which promotes resistance to chemotherapy and radiation. Fourth, Akt activation has prognostic significance for patients with NSCLC. We found that Akt activation is selective for NSCLC tumors vs. surrounding normal lung tissue and confers a poor prognosis for all NSCLC patients, but especially for those with early stage disease. This observation is highly important because most asymptomatic patients who are diagnosed through screening will have early stage disease. In 2007, we performed a series of studies in mice to show that mTOR activity is required for tobacco-carcinogen induced lung tumors. We demonstrated this by inducing lung tumors with NNK in the absence or presence of rapamycin, an FDA-approved immunosuppressant that inhibits mTOR. Rapamycin decreased tumor multiplicity by over 90% and tumor size by over 80%. This was accomplished using physiologically relevant doses of rapamycin. Subsequently, we have shown that inhibition of NNK-tumorigenesis requires depletion of lung-associated Foxp3+ cells. Foxp3+ cells are immunosuppressive, and inhibit the ability of the immune system to eliminate tumors. Thus, these studies link inhibition of the PI3K/Akt/mTOR pathway with elimination of a permissive environment that would allow lung tumors to grow without attack by the innate immune system. In the past year, we have extended these studies by investigating the efficacy of metformin in mouse models of lung cancer prevention. We chose metformin because it inhibits mTOR in diabetic patients, and diabetic patients who use metformin have a lower incidence of many cancers, including lung cancer. We showed that at steady state levels that are comparable to levels in diabetics, metformin reduces the tumor burden of NNK-induced lung tumors by over 60%. Mice had no discernible toxicities. Given these results, a clinical trial is planned in patients at risk to develop lung cancer. We have also completed studies that showed that of the three Akt isoforms, only Akt1 is required for KRas induced lung tumorigenesis. In these studies we used the NNK model as well as a transgenic mutant KRas model. In each model, loss of Akt1 but not Akt2 or 3, inhibited lung tumorigenesis by over 80%. Loss of Akt3 appeared to increase lung tumorigenesis. These studies are important because Akt inhibitors that are being tested clinically are non-isoform selective. These drugs have had only modest success, which could be related to the conflicting roles of Akt1 and 3. Our results argue that selective Akt1 inhibitors should be tested in clinical trials.